PROJECT SUMMARY Endocrine disrupting chemicals (EDCs) are a broad group of chemicals resulting in a myriad of adverse health effects. Most research to date has focused on the downstream target systems and adverse health outcomes affected by EDC exposure, but there is now increasing evidence for neuroendocrine primary targets. With the hypothalamus serving as the central regulator of multiple endocrine axes, a multitude of different specific adverse health outcomes may be observed for EDCs that target the neuroendocrine system. Atrazine, an EDC, is the second most commonly used agricultural herbicide in the United States and is the most common contaminant in potable water supplies. Atrazine is regulated by the US Environmental Protection Agency (EPA) at a concentration of 3 parts per billion (ppb; g/L) in drinking water, but concentrations above this regulatory limit are often reported. Laboratory and epidemiology studies report various endocrine disrupting and neurological adverse impacts. Most findings are focused along the hypothalamus-pituitary-gonadal axis, but studies also observe interference with the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-adrenal axes. Overall, studies support that atrazine influences multiple endocrine axes and the hypothesis of a hypothalamic toxicity target, but the mechanisms are not yet clearly elucidated and warrant further research. In addition, there are conflicting reports of the endocrine disrupting effects of atrazine near the current US EPA maximum contaminant level (MCL) of 3 ppb. Our studies using the zebrafish model system show that an embryonic atrazine exposure at concentrations ranging from 1/10X to 10X the current US EPA MCL resulted in expression alterations in genes associated with neurological and reproductive system development and function, cell cycle, and carcinogenesis; deregulation of microRNAs (miRNAs) involved with neuronal differentiation and maturation and cancer; global hypomethylation; and altered head length in larvae. Furthermore, we observed a decrease in spawning success in adults with an embryonic atrazine exposure. These adult females also had an increase in ovarian progesterone and follicular atresia and a decrease in a serotonin metabolite and serotonin turnover in the brain. Alterations in genes associated with distinct molecular pathways of the endocrine and central nervous systems were observed in brain tissue of the adult females and males. These studies support an embryonic atrazine exposure is sufficient to result in later in life adverse health outcomes associated with multiple endocrine axes. The CENTRAL HYPOTHESIS of this study is that the neuroendocrine system is the target of atrazine endocrine disruption. We seek to define the mechanisms of toxicity of an embryonic atrazine exposure that would explain the observed molecular and functional outcomes of past studies on multiple endocrine axes. We will evaluate several neuroendocrine endpoints during embryogenesis to identify disruption of the hypothalamus and/or pituitary development and neuronal structure and axonal growth alterations at the hypothalamus-pituitary interface.